The NF-κB/Rel family of transcription factors is active in inflammatory and immune cell response, cell cycle regulation, differentiation and protection from apoptosis [Baeuerle and Baltimore, Cell 87:13-20, (1996); Ghosh, et al., Annu. Rev. Immunol. 16:225-260, (1998)]. In mammals, this family of transcription factors is comprised of five members: p65 (RelA), RelB, c-Rel, NF-κB1 (which occurs both as a precursor, p105, and in a processed form, p50) and NF-κB2 (which occurs both as a precursor, p100, and as its processed product, p52). The NF-κB protein homo- and heterodimers exist in the cytoplasm, in complex with inhibitors of the IκB family. The precursor forms of NF-κB1 and NF-κB2 (p105 and p100, respectively) contain C-terminal IκB-homologous inhibitory regions. Dimers containing these NF-κB proteins are retained in the cytoplasm by virtue of the function of the IκB-homologous regions. Moreover NF-κB1/p105 and NF-κB2/p100 can also associate with dimers of other NF-κB proteins and impose cytoplasmic retention on them. NF-κB activation occurs mainly through induced degradation of the IκB proteins or of IκB homologous regions in NF-κB1/p105 and NF-κB2/p100, and consequent translocation of the NF-κB dimers to the nucleus. The induced degradation of the IκB proteins provides the most important mechanism regulating NF-κB activity (Baeuerle and Baltimore, 1996) (Ghosh et al., 1998) (Ghosh and Karin, 2002).
Most of the knowledge of these processes concerns the mechanisms of activation of a ubiquitous NF-κB dimer, p65:p50. The critical event initiating this ‘canonical’ pathway is activation of an IκB-phosphorylating protein kinase, IKK2. IKK2 occurs within a macromolecular complex, the ‘IKK signalosome’, in association with a structurally homologous kinase, IKK1, and an adapter protein, NEMO. IKK2-mediated phosphorylation of IκB leads to its proteasomal degradation and hence activation of its associated NF-κB dimers (Karin and Ben-Neriah, 2000).
Other studies have yielded some knowledge of an ‘alternative’ pathway through which NF-κB dimers containing NF-κB2/p100 are activated. This activation occurs independently of IKK2 or NEMO, but is dependent on IKK1. Phosphorylation of p100 upon activation of this pathway leads to limited proteolytic processing in which only the IκB-homologous region within p100 is degraded. This process allows the resulting p52 fragment to translocate to the nucleus in association with some other NF-κB proteins (mainly RelB) (Xiao et al., 2001) (Senftleben et al., 2001) (Solan et al., 2002) (Coope et al., 2002) (Claudio et al., 2002) (Kayagaki et al., 2002) (Dejardin et al., 2002) (Yilmaz et al., 2003) (Hatada et al., 2003).
The proteins of the tumor necrosis factor/nerve growth factor (TNF/NGF) receptor family are a group of cell-surface receptors critically involved in the maintenance of homeostasis of the immune system. These proteins interact with their corresponding ligands, either to induce cell death or promote cell survival of immune cells. The biologic function of this group of proteins has been closely associated with the regulation of the immune response and the pathogenesis of autoimmune disease. [Zhou et al., Immunol. Res. 26:323-336, (2002)]. The TNF receptors control multiple immune-defense activities as well as certain developmental processes through NF-κB activation (Wallach et al., 1999) (Locksley et al., 2001). Most of these receptors are capable of activating the canonical NF-κB pathway. In addition, the lymphotoxin-β receptor (LTβR), whose expression is restricted to stromal cells and several receptors that occur in lymphocytes (CD40, BLyS/BAFF and as shown in the present work—CD27), also activate the alternative pathway (Dejardin et al., 2002) (Coope et al., 2002) (Claudio et al., 2002) (Kayagaki et al., 2002) (Hatada et al., 2003).
Signaling for NF-κB activation by several receptors of the TNF receptor family is initiated by their binding to adapter proteins of the TRAF family. In cells treated with TNF the TRAFs have been shown to facilitate, collaboratively with the adapter protein RIP, recruitment of the signalosome components to the p55 TNF receptor (Zhang et al., 2000) (Devin et al., 2000) (Devin et al., 2001). Additional protein that participates in NF-κB activation by the TNF/NGF receptor family was identified as a ‘NF-κB-inducing kinase’ (NIK), (Malinin et al., 1997).
Initially NIK was suggested to mediate activation of the canonical NF-κB pathway in response to multiple inducers with many different physiological functions (Malinin et al., 1997). However, later studies of mice of the aly strain, which express a non-functional NIK mutant, as well as of NIK-knockout mice, challenged the notion that NIK has a functional role in the activities of most of these inducers. They suggested rather, that NIK participates selectively in the activation of NF-κB by a restricted set of ligands that specifically affect the development and function of lymphocytes (Shinkura et al., 1999) (Yin et al., 2001). Moreover, based on characterization of cells derived from these mutant mice, it was suggested that NIK does not participate at all in the canonical NF-κB pathway, but rather serves exclusively to activate the alternative one (Pomerantz and Baltimore, 2002). Lymphocytes of NIK-mutant mice exhibit a highly aberrant pattern of differentiation (Miyawaki et al., 1994) (Shinkura et al., 1999) (Matsumoto et al., 1999) (Yamada et al., 2000) (Karrer et al., 2000) (Fagarasan et al., 2000), therefore, the present work aimed to re-assess the signaling role of NIK in lymphocytes.
In the present, the function of NIK in lymphocytes was now re-evaluated by assessing the effect of its depletion or inhibition in vitro in cultured cells of lymphoblastoid lines. The assays showing that NIK is not required for activation of the canonical pathway by TNF in lymphocytes were confirmed. However, as detailed below, NIK was found to play a crucial role in these cells in activation of the alternative as well as of the canonical pathway by CD40 ligand (CD40L) and BLyS/BAFF induction. Furthermore, CD27 (Camerini et al., 1991), a receptor of the TNF/NGF family that is expressed mainly in T lymphocytes and memory B lymphocytes and was previously suggested to activate NF-κB (Yamamoto et al., 1998) in a NIK-independent manner (Akiba et al., 1998) was shown to initiate the alternative pathway. I was also found by the inventors that NIK binds to SIVA, a protein associated with CD27 (Prasad et al., 1997), and mediates both the canonical and the alternative NF-κB-activating pathways in response to this receptor. Although NIK was not required for activation of the signalosome by the p55 TNF receptor, activation of the signalosome by CD27 did depend on NIK. Moreover, unlike triggering by the p55 TNF receptor, triggering by CD27 induced, in a NIK-dependent way, selective recruitment of IKK1 to this receptor, a process that might be the initiating event in the NIK-dependent activation of both NF-κB pathways by CD27.
The biologic function of members of the NIK-dependent NF-κB pathway has been closely associated with the regulation of the immune response and the pathogenesis of autoimmune disease.
It is shown in accordance with the present invention that NIK, in contrast to prior art teachings, does participate in the canonical NF-κB activating pathway. In addition, it is shown that NIK participates in an alternative NF-κB pathway which is induced by BlyS and CD40L and have identified CD70 as a novel inducer of this alternative pathway.
As such, the present findings establish the role of NIK in NF-κB activation and thus provide the motivation to utilize various NIK targeting agents in treatment of various immune diseases.